The invention relates to the field of cell and tissue culture with the help of a culture fluid or nutrient medium.
This invention more particularly relates to devices and methods for cell and tissue culture in which the culture fluid (or nutrient) is set into motion so as to achieve a dynamic culture. Such devices usually apply either a technique for directly stirring the culture fluid in the culture volume or a technique providing permanent flow of the fluid through a specific circuit.
These techniques for fluid flow are difficult to control because culture conditions change over time. Actually, the low cell concentration which exists at the beginning of the culture process, requires a quasi-static environment, and therefore a very low culture fluid flow rate, whereas cell multiplication or tissue growth needs a rapidly increasing flow rate.
Maintaining adequate conditions during the whole culture process requires either the use of several suitable devices, at different stages of the culture""s growth, respectively, and this involves many handling operations, or complex and often cumbersome means, which limit, even precludes observation of the culture""s development with a microscope. These handling operations and means are all the more complex as the culture process should be carried out in a sterile environment which requires highly qualified personnel, and this therefore increases the cost for each culture. Furthermore, because of their price, these devices should be reusable, which imposes complete sterilization before each new culture, and maintenance operations.
The object of the invention is therefore to overcome all or part of the aforementioned drawbacks.
For this purpose, it provides a cell and tissue culture device which initially comprises a pressurization circuit capable of delivering at least a selected gas under a selected pressure, at least a culture chamber accommodating supporting means for at least a deformable membrane forming inside the chamber an interface between first and second portions with variable complementary volumes and supplied with culture fluid by a first tank and with selected gas by the pressurization circuit, respectively; the first portion further receiving the cells or tissues to be grown. The device further comprises control means capable of determining, according to selected criteria relating to the type of culture to be achieved, the gas to be fed into the second portion, its pressure and the time for feeding it in, and of controlling access to the first and second portions of the chamber so as to control together the shape of the membrane, culture fluid supply and flow of this fluid in the first portion.
By varying the pressure of the gas in the second portion, the shape of the membrane(s) may be changed, and the culture fluid may be forced to flow in the first portion of the chamber. For example, overpressurization phases may be alternated with depressurization phases in the second portion of the chamber in order to cause oscillation of the membrane(s).
Thus, for example, defining for each type of culture, a series of parameter n-tuples each including at least a selected gas, a pressure for this gas, a time for feeding in this gas, a culture fluid flow rate and a time for feeding in this fluid, is sufficient for controlling the growth of this culture. However, more complex modes may be considered which are based on adapting the multiple parameters from physical measurements, such as temperature measurements, or measurements of the proportion of molecular species or even pH measurements, carried out in the chamber and/or in the pressurization circuit.
All these parameters may be stored and/or calculated in the control means. They may be also changed through reprogramming.
Conducting pressure measurements in the culture chamber, or measurements of culture fluid level in the culture chamber or even calorimetric measurements for quantifying growth of certain cultures, or even redox potential measurements may also be considered.
In the case of a single membrane device, providing a supramembrane above the latter may be advantageous, in order to delimit between them at least an intermediate area supplied with gas by the pressurization circuit and able to discharge at least a portion of this gas, wherein the control means are configured so as to determine, according to selected criteria, the gas to be fed into the intermediate area, its pressure and time for feeding it in, and for controlling access to the inlet and outlet of the intermediate area so as to control together the shapes of the membrane and supramembrane and culture fluid flow in the first portion. This supramembrane may be firmly secured to the membrane at a multiplicity of selected locations, so as to subdivide the intermediate area into a plurality of communicating cellular cavities of variable volumes.
The intermediate area may optionally be fed with a gas other than the one fed into the second portion.
In a preferred embodiment of the device according to the invention, supporting means are configured so as to support a multiplicity of independent membranes, which may be gathered together in two, three, four or even more independent groups. Thus, according to the type of programming for the control means and according to the number of membrane groups, it is possible to generate a large number of fluid flow modes in the first portion, for example, an essentially xe2x80x9chorizontalxe2x80x9d flow, an essen-tially xe2x80x9cverticalxe2x80x9d flow, a combination of vertical and horizontal flows, or even a xe2x80x9czig-zagxe2x80x9d flow.
The number of flow modes may further be increased by providing the first portion of the culture chamber on the opposite side of the second portion, with a deformable pocket or one or more auxiliary deformable membranes supplied with gas, preferably with the selected gas by the pressurization circuit. However, it may be a different complementary gas, (in this case, it is clear that the pressurization circuit should include two parallel branches supplied with two different gases).
If several auxiliary membranes are used, they may be subdivided into independent groups (two, three, four or more), as previously explained for the other membranes. Each group may also be subdivided into independent subgroups, subject to adaptation of the gas supply mode for the groups (addition of supply ducts and valves).
Moreover, and always in this case, partitioning means may be provided in the central part of the first portion, defining within the latter, culture cavities each extending from a group of membranes to a group of auxiliary membranes, wherein each cavity is supplied with culture fluid. These partitioning means may include for example partitions made of a porous material to the culture fluid, but impervious to cells and tissues, so that only the culture fluid flows from one cavity to another.
On the other hand, the first portion may accommodate at least a deformable side membrane forming an interface between a side portion and the portion containing the cells or tissues, wherein the side portion is supplied with gas by the pressurization circuit and may discharge at least a portion of this gas. Of course, in this case, the control means are configured so as to determine, according to selected criteria, the pressure and the time for feeding in the gas, and for controlling access to the inlet and outlet of the side portion in order to control the shape of the side membrane(s). The gas introduced into this side portion may either be the gas selected for feeding the second portion, or another selected gas (with an additional branch in the pressurization circuit).
Preferably, each membrane, side membrane, auxiliary membrane and pocket is made of a porous material, at least in the direction pointing to the first portion which contains the cells or tissues to be grown, so that the selected gas may at least partially penetrate the first portion.
The device may comprise several culture chambers, for instance two or three, or even more. These chambers may be accommodated in a compartment and/or be external, whereby their connection to the pressurization circuit is achieved preferably via an interface.
The device according to the invention may include additional characteristics considered separately or combined and notably:
the chamber is preferably delimited by a case comprising a lower portion forming a receptacle and an upper portion forming a lid, preferably transparent, wherein the supporting means comprises a frame accommodated within the receptacle and wherein appropriate cavities are defined, each for sealably supporting a membrane, to be supplied with selected gas and to accommodate this membrane when the pressure of the selected gas is lower than a threshold, and the frame is preferably made out of a synthetic material, particularly an elastomer;
each inlet and each outlet is advantageously controlled by access control means driven by control means;
each culture chamber may be connected to a first tank containing the culture fluid and a second tank for collecting the waste culture fluid, and form an autonomous culture unit; each tank preferably comprising at least a port provided with sealing means, particularly with a septum type, allowing fluid or any other body to be introduced therein or extracted therefrom;
the compartment accommodating the control means and the pressurization circuit preferably comprises a sub-compartment forming a gas tank supplied with selected gas by the supplied inlet(s) and feeding the compressor; wherein the pressurization circuit then preferably includes a duct connecting an outlet of the sub-compartment to the inlet of the pocket or of the rear portion of the first portion, delimited by the auxiliary membrane(s);
the sub-compartment may be subdivided into at least first and second sealed sub-portions forming a first low pressure gas tank, supplied with selected gas by each supply inlet and feeding the compressor, and a second high pressure gas tank fed by the compressor and supplying each outlet of the pressurization circuit with selected gas, respectively. A third sub-portion may also be provided between the first and second sub-portions so as to form a third intermediate pressure gas tank, wherein the different sub-portions communicate which each other through ports, access to which is controlled by control means, and each sub-portion may be supplied with selected gas, under the control of access control means driven by control means, each outlet of the pressurization circuit.
The invention also relates to a method for growing cells and tissues. This method notably consists of:
a first step in which a culture device is provided, comprising at least a culture chamber provided with at least a deformable membrane at the interface between first and second portions with variable complementary volumes, wherein the first portion is able to be fed with a culture fluid and to receive cells and/or tissues to be grown, and the second portion is able to be supplied with gas; and
a second step wherein the first and second portions are fed according to pressures, flow rates and times selected depending on criteria relating to the type of culture to be achieved, in order to change the shape of the membrane(s) during the culture period, so as to create a controlled flow of the culture fluid in the first portion of the chamber.
Preferably, in the first step, a chamber is provided, comprising a multiplicity of independent membranes, wherein more preferably these are gathered together in two, three or four groups of independent membranes, or even more, and in the second step, each group is independently supplied with selected gas so as to independently change the relevant shapes of the membranes of each group.
Also preferably, in the first step, it is expected that the first portion of the culture chamber comprises, on the opposite side to the second portion, a deformable pocket or one or more auxiliary membranes supplied with gas, preferably with the selected gas, and in the second step, the pocket or the membrane(s) are fed according to selected pressure, flow rates and times depending on the criteria, so as to change the shape of this pocket together with that of each membrane during the culture period. Advantageously, each membrane and the pocket or the auxiliary membrane are made of a porous material, at least in the direction pointing to the first portion, so that the selected gas, introduced in the second portion and/or the pocket or the auxiliary membrane, may at least partially penetrate the first portion.
Thus, as shown for the device, according to applied pressures, times and flow rates and according to the number of membrane groups used, it is possible to generate a large number of fluid flows in the first portion.